Process for preparing mouse interferon

ABSTRACT

The present invention relates to a process which is easily applicable for industrial production of mouse interferon. 
     Particularly, the present invention relates to a process based on the discovery that a large amount and high activity of mouse interferon is obtained by transplanting established mouse tumor cells to other warm-blooded animal body or inoculating the cells in a culture medium charged in a filter-membrane-interposed diffusion chamber which is designed and fitted up to or in the animal body so that its nutrient body fluid feeds the cells, multiplying the transplanted or inoculated cells in the warm-blooded animal body or the diffusion chamber utilizing said body fluid, and exposing the multiplied cells to the action of interferon inducer in vitro or in vivo.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for preparing mouse interferon.

As reported in "Science", vol. 177, pp. 797-799 (1972), it is well documented that normal mouse cells induce mouse interferon of relatively high activities, while mouse tumor cells induce mouse interferon of considerably low activities. Normal mouse cells are, however, not proper for large-scale production of mouse interferon because they multiply much slower than mouse tumor cells.

Envisaging the utilization of high multiplication rates of mouse tumor cells, the present inventors performed studies for large-scale production of mouse interferon with said cells.

The efforts resulted in the discovery that a large amount of and high-titred mouse interferon was not obtainable by inoculating and culturing established mouse tumor cells in a culture medium in vitro, or transplanting and multiplying the cells in mouse body utilizing its nutrient body fluid, but easily obtainable by transplanting said cells in other warm-blooded animal body or inoculating the cells in a culture medium charged in a filter-membrane-interposed diffusion chamber which is designed and fitted up to or in the animal body so that its nutrient body fluid feeds the cells, multiplying the transplanted or inoculated cells in the warm-blooded animal body or the diffusion chamber utilizing said body fluid, exposing the multiplied cells to the action of interferon inducer in vitro or in vivo to induce a high-titred mouse interferon, and purifying and separating the induced mouse interferon.

The process according to the present invention differs from the conventional processes in which the cells are multiplied in vitro, and has advantages that it requires no or much less nutrient medium supplemented with expensive serum, that the maintenance and control of the conditions during the multiplication of the established mouse tumor cell are much easier, and that higher-titred mouse interferon is easily obtainable.

More particularly, in the process according to the present invention, established mouse tumor cells can be easily multiplied in other warm-blooded animal body utilizing the body fluid by either transplanting said cells therein, or embedding or connecting in or to the animal body with a diffusion chamber charged with a culture medium suspended with said cells, while feeding the animal in the usual way.

As compared with the conventional processes in which established mouse tumor cells are multiplied in vitro, the process according to the invention in which the cells are multiplied utilizing nutrient body fluid from the warm-blooded animal body has further features that the multiplication of the cells is steadier, the multiplication rate is higher, and that the yield of the induced interferon per cell is much higher.

Any established mouse tumor cell may be used as far as it can multiply when transplanted in other warm-blooded animal body; for example L 5178 Y cell, SM 36 cell, L 1210 cell, FAC-C cell, T 3 cell, M 1 cell, OUMS-2 cell, JTC-11 cell, ELD cell and Sarcoma 180 cell, as reported in "Protein, Nucleic Acid and Enzyme", vol. 20, no. 6, pp. 616-643 (1975). Particularly, mouse lymphoblastoid cell lines such as L 5178 Y cell, L 1210 cell, OUMS-2 cell and JTC-11 cell are preferable because of their high multiplication rates and the attainment of high activities of the induced interferon.

Any warm-blooded laboratory animal can be used in the invention as far as the transplanted established mouse tumor cells can multiply therein; for example birds such as chicken and pigeon, and mammalians such as dog, cat, monkey, goat, pig, bovine, rabbit, horse, guinea pig, rat and hamster.

Since transplantation of mouse tumor cells in the above-mentioned animal body tends to cause undesirable immunoreactions, animals in the most immature state, namely, egg, fetus, embryo, or new-born or infant animal, should be chosen to depress the immunoreactions as much as possible.

Prior to transplantation of the cell, the animal may be pretreated, for example either by irradiating with about 200-600 rem of X-ray or γ-ray, or with antiserum or an immunosuppressive agent, to depress the immunoreactions.

Instead of transplanting and multiplying established mouse tumor cells in animal body, the cells can be inoculated and multiplied in a nutrient of other warm-blooded animal body in a conventional-type diffusion chamber which is embedded, for example intraperitoneally, and devised to allow the cells to utilize said body fluid.

The chambers which are usable in the invention can be of various shapes and sizes and should be interposed with filter membranes, for example membrane filter, ultra-filter and hollow fiber, to prevent the leakage of the cells. Particularly, the chambers with interposed filter membranes of pore sizes of about 10⁻⁷ to 10⁻⁵ m are preferable.

In addition, established mouse tumor cells, if necessary, can be multiplied in a nutrient body fluid circulating through the diffusion chamber which is connected to a certain part of the animal body and placed, for example on the surface of the animal body, to allow the animal to feed the cells with its nutrient body fluid.

Furthermore, designing and connecting the diffusion chamber so that it can be disconnected periodically from the animal body and the cells multiply through the whole life of the animal without any unnecessary sacrifice of animal, the animal will further increase the yield of the multiplied cells per animal.

Furthermore, the process using the above-mentioned diffusion chamber has an additional feature besides the multiplied mouse tumor cells can be easily harvested because there is no direct contact of the cell with the animal cell, that various warm-blooded animals can be used without any pretreatment to depress their immunoreactions because of their lower possibilities of causing the immunoreactions.

As to the induction of mouse interferon, any method can be employed as far as it induce interferon in the multiplied living mouse cells.

The multiplied cells can be exposed in vivo to the action of interferon inducer wherein they developed, or exposed in vitro to the action of interferon inducer after isolation of them.

Particularly, when mouse tumor cells are multiplied in a diffusion chamber, the exposure of the multiplied cells to the action of interferon inducer in or out of the chamber induces mouse interferon.

Any known interferon inducer can be used as far as it induce mouse interferon; for example virus, bacterium, protozoon, rickettsia, nucleic acid, endotoxin and polysaccharide.

The induced mouse interferon can be readily purified, separated and collected by conventional purification and separation techniques, for example salting out, dialysis, filtration, centrifugation, concentration and freeze-drying.

If more highly purified mouse interferon preparations are desirable, mouse interferon of the highest purity will be obtainable by employing conventional techniques, for example adsorption on and desorption from ion exchanger, gel-filtration, affinity-chromatography, isoelectric point fractionation and electrophoresis, in combination with the above-mentioned techniques.

In the present invention, the interferon activity was determined by the plaque reduction method described by R. R. Wagner, "Biological Studies of Interferon. Virology", vol. 13, pp. 323-337 (1961), in which mouse L cells were challenged to vesicular stomatitis virus.

The hemagglutination titer was assayed according to the method described by J. E. Salk, "Journal of Immunology", vol. 49, pp. 87-98 (1944).

Several embodiments for preparing mouse interferon according to the invention are described below.

EXAMPLE EXAMPLE 1

New-born hamsters were pre-injected with antiserum prepared from rabbit according to the known method and then were subcutaneously transplanted with established mouse tumor cells of L 1210 cells. The hamsters were fed in the usual way for 3 weeks.

The subcutaneously-occurred massive tumors were isolated, cut finely and dissociated in a physiological saline solution containing trypsin at 4° C. The cells were washed with an Eagle's minimal essential medium supplemented with 5 v/v % of fetal bovine serum at pH 7.2 and 37° C., and were resuspended in the same medium to give a concentration of about 1×10⁷ cells per ml. Then, to the suspension was added partially-purified mouse interferon in a proportion of about 50 units per ml. The mixture was incubated at 37° C. for about 6 hours.

Then, the mixture was added with Newcastle disease virus in a proportion of about 300 of hemagglutination units per ml and incubated for an additional 20 hours to induce mouse interferon.

The incubated culture was centrifuged at about 1,000×g and about 4° C. to remove precipitates such as cell residues. The resulting supernatant was first dialyzed against 0.1 M hydrochloric acid-potassium chloride buffer at pH 2.0 and 4° C. for 70 hours, and then dialyzed against a physiological saline solution bufferized at pH 7.2 with 0.01 M phosphate buffer, for 12 hours. The dialyzed mixture was carefully filtrated with membrane filter and concentrated to obtain an interferon solution.

The resulting solution was used in the following experiments as partially-purified interferon solution.

As a control experiment, the tumor cells which were similarly multiplied by transplanting L 1210 into nice were treated as in the case of hamster to induce interferon. The obtained interferon was partially purified.

The activities of the interferon preparations obtained respectively by the present invention and control experiment before and after the partial purification are shown in Table 1.

As obvious from the results shown in Table 1, extremely high-titred mouse interferon is induced in the mouse tumor cells which are multiplied in hamster, and the partial purification of the induced mouse interferon using hamster results in a 100 times or more higher activity than that of the control preparation.

                  TABLE 1                                                          ______________________________________                                                         present invention                                                                             control                                         animal          hamster        mouse                                           ______________________________________                                         activity of induced                                                            interferon (units/ml)                                                                          27,000         600                                             activity of partially-                                                         purified interferon                                                            (units/animal)  18,000,000     110,000                                         ______________________________________                                    

EXAMPLE 2

New-born hamsters were pre-injected with antiserum to depress their immunoreactions and were intraperitoneally transplanted with established mouse tumor cells of JTC-11 cells. The hamsters were fed in the usual way for 4 weeks.

The cells were harvested from about 7 ml of the ascites per hamster and treated similarly as described in Example 1 to obtain a partially-purified and concentrated mouse interferon.

The interferon activity of the concentrate was about 1,700,000 units per hamster.

EXAMPLE 3

Adult hamsters were irradiated with about 300 rem of γ-ray to depress their immunoreactions and intraperitoneally transplanted with established mouse tumor cells of OUMS-2 cells. The hamsters were fed in the usual way for 3 weeks.

The multiplied cells were collected from the occurred massive tumors and mouse interferon was induced, partially purified and concentrated, similarly as described in Example 1. Then, the mouse interferon-containing concentrate was freeze-dried into powder.

The interferon activity of the powder was about 9,400,000 units per hamster.

EXAMPLE 4

After the rats were irradiated with about 400 rem of X-ray to depress their immunoreactions, the rats were intraperitoneally transplanted with established mouse tumor cells of L 5178 Y cells and were fed in the usual way for 2 weeks.

Two days after intraperitoneally injecting about 5,000 hemagglutination units of Newcastle disease virus whose activity was almostly inactivated, the rats were sacrificed to harvest the ascites. The ascites was treated in the similar way as described in Example 1 which resulted in the obtainment of a partially-purified interferon solution with an interferon activity of about 300,000 units per rat.

EXAMPLE 5

Using plastic cylindrical diffusion chambers, capacities of about 20 ml, with interposed membrane filters having pore sizes of about 0.5μ, established mouse tumor cells of OUMS-2 cells were suspended in physiological saline solution. Two chambers were intraperitoneally embedded in one dog.

The dogs were fed in the usual way, and about 4 weeks later the chambers were removed from the dogs.

The cell concentration of the chambers was about 4×10⁹ cells per ml which was about 10² to 10³ times or more higher than that attained in vitro on a nutrient medium in a CO₂ incubator.

The cells were treated and the induced mouse interferon was partially purified and concentrated, similarly as described in Example 1. Then, the mouse interferon-containing concentrate was freeze-dried into powder.

The interferon activity of the powder was about 50,000,000 units per dog.

EXAMPLE 6

Established mouse tumor cells of JTC-11 cells were transplanted in the allantoic cavities of embryonated eggs which had been pre-incubated at 37° C. for 5 days, and then were incubated for an additional 5 days.

The eggs were opened and the multiplied mouse tumor cells were harvested. Then, the cells were treated and the induced mouse interferon was partially purified and concentrated, similarly as described in Example 1.

The interferon activity of the concentrate was about 700,000 units per 10 embryonated eggs. 

What we claim is:
 1. In the process for producing mouse specific interferon comprising cultivating established mouse cells, exposing the resultant cells to the action of an interferon inducer to induce interferon, and collecting and purifying the interferon, the improvement wherein the established mouse cells are established mouse lymphoblastoid cell lines selected from the group consisting of L 5178 Y cell, L 1210 cell, OUMS-2 cell and JTC-11 cell, and wherein said cultivating step comprises transplanting the established mouse tumor cells into another non-mouse warm-blooded laboratory animal body, and wherein said exposing step is in vivo or in vitro.
 2. A process according to claim 1 in which the non-mouse warm-blooded laboratory animal is a mammalian.
 3. A process according to claim 1, wherein said non-mouse warm-blooded laboratory animal is a bird, dog, cat, monkey, goat, pig, bovine, rabbit, horse, guinea pig, rat or hamster. 